Reconstruction of mitochondrial cytochrome P450-dependent steroid hydroxylases in artificial phospholipid membranes: studies of cytochrome P450scc topology by limited proteolysis.
Highly purified cytochrome P450scc from bovine adrenal cortex mitochondria was inserted in artificial phospholipid membranes prepared from phosphatidylcholine to study the main principles of its membrane organization in the model system. Topology of the cytochrome P450scc polypeptide chain in proteoliposomes was studied by limited proteolysis with trypsin or chymotrypsin followed by immunochemical identification of the products of proteolysis products of the membrane-bound heme protein. It is shown that limited proteolysis of cytochrome P450scc in proteoliposomes results in a significant decrease of Vmax for the reaction of cholesterol hydroxylation to pregnenolone in the reconstituted system in the presence of exogenously added adrenodoxin-reductase and adrenodoxin. However, after proteolytic modification of cytochrome P450scc with trypsin and chymotrypsin the affinity of the heme protein to adrenodoxin is increased. Different models of membrane organization as well as functional specificity of cytochrome P450scc in artificial membranes are discussed.
Krivosheev
A V
AV
Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, Minsk, Belarus. usanov@ns.iboch.ac.by
Usanov
S A
SA
D000302:Adrenal Cortex / Q000201:enzymology*; D000325:Adrenodoxin / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D011284:Pregnenolone / Q000378:metabolism; D012995:Solubility; D013053:Spectrophotometry; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
D008081:Liposomes; D008565:Membrane Proteins; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D000325:Adrenodoxin; D011284:Pregnenolone; D002784:Cholesterol; D013250:Steroid Hydroxylases; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase; D002918:Chymotrypsin; D014357:Trypsin